Synergistic antibacterial compositions and method of treatment of infections caused by multiple antibiotic-resistant organisms

ABSTRACT

Antibiotic compositions comprising a 1-oxa β-lactam antibiotic compound of the formula ##STR1## or pharmaceutically acceptable salts thereof and either tobramycin, amikacin or piperacillin exhibit synergistic activity against multiple-antibiotic-resistant organisms. The 1-oxa compound can be used in conjunction with tobramycin, amikacin or piperacillin in a method of treating infections caused by resistant organisms.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 332,774, filed12-21-81 now U.S. Pat. No. 4,452,778 issued June 5, 1984, which was acontinuation-in-part of application Ser. No. 135,390, filed Mar. 31,1980, now abandoned, which was a continuation-in-part of applicationSer. No. 36,263, filed May 4, 1979, now abandoned.

BACKGROUND AND SUMMARY OF THE INVENTION

This invention is directed to antibiotic compositions exhibitingsynergistic activity against a number of multiple-antibiotic-resistantorganisms, and to a method of treating infections caused by suchorganisms in man and other warm-blooded animals. More particularly thisinvention is concerned both with synergistic antibiotic compositionscontaining moxalactam, the 1-oxa β-lactam antibiotic compound of theformula ##STR2## or pharmaceutically acceptable salts thereof and,either tobramycin, amikacin or piperacillin (sodium6-(D(-)-α-(4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino)-α-phenylacetamido]penicillinate),and with a method of treating infections utilizing the 1-oxa antibioticcompound I in combination with tobramycin, amikacin or piperacillin.

The mutual effect of simultaneously administered antibiotics, exerted oneach other and on various pathogenic microorganisms, has been thesubject of much research. The literature is replete with reports byinvestigators whose experiments have shown unambiguously that amongpreviously known antibiotics, either synergism or antagonism may occur.In the case of synergism the antibiotic combination exhibits a markedincrease in activity over that which could be predicted as the result ofa purely additive effect of the two or more drugs in combination. Bothquantitative and qualitative synergistic effects have been observed.

The treatment of infections due to multiple-antibiotic-resistantorganisms presents a challenge which a number of clinicians have in thepast sought to meet through the utilization of synergistic antibioticcombinations. The lower effective minimum inhibitory concentrations(mics) of synergistic combinations of the present invention as well asothers against such organisms allow for the treatment of those moredifficult infections at lower dosage levels than otherwise possible,thereby lowering the probability of toxicity complications, the time fortreatment, and, potentially, the cost of therapy. The present inventiontherefore constitutes a significant addition to the physician'smedicinal armamentarium.

The antibiotic compounds utilized in the present invention are knowncompounds. Tobramycin and amikacin or their sulfuric acid addition saltsare commercially available broad spectrum aminoglycoside antibiotics.Piperacillin is a relatively new penicillin antibiotic compound. Itspreparation is described by I. Saikawa et al. in the Journal of thePharmaceutical Society of Japan, 597, No. 9, p 980-986 (1977). The 1-oxaβ-lactam compound designated by formula I above was recently describedin U.S. Pat. No. 4,138,486, issued Feb. 6, 1979.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that the 1-oxa β-lactamantibiotic compound I when combined with either tobramycin, amikacin orpiperacillin exhibits synergistic antibiotic activity against a numberof strains of multiple-antibiotic-resistant microorganisms. Using thebroth dilution technique in a checkerboard distribution it wasdetermined that the 1-oxa compound I when combined with tobramycinexhibited mics of about 0.012 to about 4 mcg/ml against strains ofPseudomonas aeruginosa, Serratia marcescens, E. coli, E. cloacae,Klebsiella pneumoniae, Proteus rettgeri, Proteus mirabilis, Proteusvulgaris, Pseudomonas maltophilia, and Acinetobacter sp. Similarly itwas determined that the 1-oxa compound I when combined with amikacinexhibited mics of about 0.06 to about 8 mcg/ml against strains ofProteus rettgeri, Klebsiella pneumoniae, Serratia marcescens, E.cloacae, Pseudomonas aeruginosa, Pseudomonas maltophilia, Pseudomonaspseudomallei and Acinetobacter sp. It was also determined that the 1-oxacompound I when combined with piperacillin exhibited mics of about 0.12to about 16 mcg/ml against strains of Pseudomonas aeruginosa, Serratiamarcescens, E. coli, and acinetobacter sp. Synergistic activity againstPseudomonas aeruginosa strains is especially notable in combinations ofthe 1-oxa compound and tobramycin or piperacillin.

Clinical advantage of the observed synergistic activity can be realized,therefore, by administering the 1-oxa compound I or a pharmaceuticallyacceptable salt thereof in conjunction with either tobramycin, amikacinor piperacillin at dosages that will provide synergistically therapeuticblood levels of each antibiotic. Generally speaking it can be said thatagainst a given organism the therapeutic level of a compound in theblood is that level equal to or greater than the minimum inhibitoryconcentration measured for that compound by in vitro studies. To assurethe efficiency of the 1-oxa compound when used in combination withtobramycin, amikacin or piperacillin in the method of this invention,minimum blood levels for the 1-oxa compound are preferably at leasttwice the in vitro-measured combination mic for that compound.

Thus one feature of the present invention is a method of treatinginfections caused by multiple-antibiotic resistant Gram-negativeorganisms in a warm-blooded animal which comprises administering to saidanimal the 1-oxa compound I or a pharmaceutically acceptable saltthereof in an amount sufficient to produce blood levels of said compoundin said animal of at least about 0.02 to about 8 mcg/ml; andadministering in conjunction therewith a synergistically effectiveamount of tobramycin.

A further embodiment of the present invention is a method of treatinginfections caused by multiple-antibiotic resistant Gram-negativeorganisms in a warm-blooded animal which comprises administering to saidanimal the 1-oxa compound I or a pharmaceutically acceptable saltthereof in an amount sufficient to produce blood levels of said compoundin said animal of at least about 0.12 to about 16 mcg/ml; andadministering in conjunction therewith a synergistically effectiveamount of amikacin.

Another embodiment of the present invention is a method of treatinginfections caused by multiple-antibiotic-resistant Gram-negativeorganisms in a warm-blooded animal which comprises administering to saidanimal the 1-oxa compound I or a pharmaceutically acceptable saltthereof in an amount sufficient to produce blood levels of said compoundin said animal of at least about 0.2 to about 32 mcg/ml; andadministering in conjunction therewith a synergistically effectiveamount of piperacillin.

The term "multiple-antibiotic-resistant Gram-negative organisms" as usedherein refers to those Gram-negative organisms that have shownresistance to several classes (for example, penicillins, cephalosporins,aminoglycosides, macrolides etc.) of antibiotic compounds.

A "synergistically effective amount" as used in the foregoingdescription of the method embodiments of the present invention refers tothat amount of antibiotic (piperacillin, amikacin or tobramycin) whichwill on administration provide concentration of that antibiotic in theblood equal to or above the minimum concentration necessary forsynergistic co-action of that antibiotic and the 1-oxa compound at thelevel administered in conjunction therewith. In this regard it should benoted that results of combination synergy studies are reported in termsof minimum inhibitory concentrations of each compound when tested incombination with the others. These values are regarded simply as anindicator of the presence (or absence) of synergistic co-action of thecombined antibiotics. If synergy is in fact indicated by the combinationmics, the combined antibiotics will usually display synergistic activityover a wide range of concentrations of each antibiotic. Analogouslysynergistic activity can be exploited in vivo over a wide range of bloodserum levels for each antibiotic. Thus, for example, for any given bloodlevel, equal to or above about twice the in vitro 1-oxacompound-tobramycin combination mic of the 1-oxa compound, clinicaladvantage of the synergistic activity can be had over a range oftobramycin blood levels.

The dosage required of any of the antibiotic compounds utilized in thepresent method to achieve the desired synergistically therapeutic bloodlevels depends on a number of factors including animal or patientweight, the nature of the infection, the particular method ofadministration, and the pharmacokinetics of the particular antibiotics.

The pharmacokinetics or more particularly the blood level-dose responsefor tobramycin is well documented. [See for example the Physicians' DeskReference, 33rd Edition, published 1979, by Medical Economics, Inc., adivision of Litton Industries, Inc., Oradel, N.J., pages 1043-1045].Following an intramuscular dose of 1 mg/kg of tobramycin sulfate maximumserum concentrations reach about 4 mcg/ml. For example, peak serumconcentrations of tobramycin in healthy volunteers receiving a single 1mg/kg (i.m.) were 4.4 mcg/ml. Single doses of tobramycin (sulfate) forthe present method range from about 0.6 to about 2 mg/kg and morepreferably from about 0.6 to about 1.5 mg/kg. Such dosages provide bloodlevels of tobramycin at synergistically effective concentrations ofbetween at least about 0.5 mcg/ml to about 8 mcg/ml.

The blood level-dose response for amikacin is likewise well-documented.See the Physicians' Desk Reference, 34th Edition, published 1980, byMedical Economics, Inc., a division of Litton Industries, Inc., Oradel,N.J., pages 701-703. Average peak serum concentrations of about 12, 16and 21 mcg/ml are obtained 1 hour after intramuscular administration of250 mg (3.7 mg/kg), 375 mg (5 mg/kg), 500 mg (7.5 mg/kg) single dosesrespectively. At 10 hours serum levels are about 0.3, 1.2 and 2.1 mcg/mlrespectively. Single doses of 500 mg (7.5 mg/kg) administered to normaladults as an infusion over a period of 30 minutes produced a mea peakserum concentration of amikacin of 38 mcg/ml at the end of the infusionand levels of 24 mcg/ml, 18 mcg/ml and 0.75 mcg/ml after 30 minutes, 1hour and 10 hours post-infusion respectively. Single doses of amikacin(sulfate) for the present method range from about 1 mg/kg to about 6mg/kg and more preferably from about 1 mg/kg to about 3 mg/kg with theparticular dose being dependent on patient weight, the infectingorganism and the method of administration. According to this embodiment,synergistically effective blood levels of amikacin of about 0.5 mcg/mlto about 8 mcg/ml are provided by the above dosages.

Several investigators have studied extensively the pharmacokinetics ofpiperacillin [M. A. Evans et al., Journal of Antimicrobial Chemotherapy,4, 255-261 (1978) and T. B. Tjandramaga et al., Antimicrobial Agents andChemotherapy, Vol. 14, No. 8, 829-837 (1978)]. Single intramusculardoses of 0.5, 1.0, and 2.0 g of piperacillin resulted in mean peak serumconcentrations of 4.9, 13.3 and 30.2 mcg/ml respectively. Singleintravenous bolus doses of 1.0, 2.0, 4.0 and 6.0 g of piperacillinprovided mean serum concentrations of 70.7, 199.5, 330.7 and 451.8mcg/ml respectively at the end of the injections. The antibiotic has amean terminal serum half-life of 60 to 80 minutes after theintramuscular doses and 36 to 63 minutes after the intravenousadministration depending on the dose. Single doses of piperacillin forthe present method range from about 250 mg to about 4 g and morepreferably from about 250 mg to about 2 g.

Serum level response to various intravenous doses of the 1-oxa compoundI is summarized in Table I.

                                      TABLE I                                     __________________________________________________________________________    MEAN SERUM CONCENTRATIONS* OF 1-OXA COMPOUND I                                Time in hours                                                                 Dose**                                                                              1/6 1/3 1/2 1  2  3  5  7 9 11                                                                              12                                        __________________________________________________________________________    250 mg IV                                                                           10.9                                                                              19.8                                                                              17.7                                                                              16.4                                                                             12.4                                                                             6.3                                                                              3.2                                                                              1.5                                                                             1.4                                           500 mg IV                                                                           17.1                                                                              28.4                                                                              27.2                                                                              22.9                                                                             16.9                                                                             7.5                                                                              4.6                                                                              2.6                                              1 gm IV                                                                            34.9                                                                              68.2                                                                              46.6                                                                              24.7                                                                             20.5                                                                             15.6                                                                             8.9                                                                              6.1                                                                             1.5                                                                             1.2                                          2 gm IV                                                                            101.8                                                                             152.2                                                                             135.8                                                                             89.2                                                                             48.7                                                                             31.2                                                                             26.1                                                                             8.7                                                                             4.0                                           __________________________________________________________________________     *mcg/ml                                                                       **Infused over 20 minute period                                          

Thus a 250 mg intravenous dose of the 1-oxa compound will provide serumlevels of from about 10 to about 20 mcg/ml for a 2 hour period. Theminimum serum levels of the 1-oxa compound I necessary for synergisticactivity according to the present method range from about 0.02 mcg/ml toabout 32 mcg/ml. These minimum serum levels can be easily achieved andeven exceeded by the administration of single doses of about 250 mg toabout 2 g and more preferably about 250 mg to about 1 g of the 1-oxacompound I. Those skilled in the art can optionally adjust the dose ofthe 1-oxa compound below or above these levels in accordance with theirevaluation of each clinical case. According to this embodiment,synergistically effective blood levels of piperacillin are between about0.5 mcg/ml to about 32 mcg/ml.

In carrying out the present method two antibiotics are administered to awarm-blooded animal in conjunction with one another to achieve apotentiated antibiotic effect. The antibiotic compounds can beadministered intramuscularly or by intravenous infusion of bolusinjection, either sequentially or simultaneously through the same ordifferent routes of administration.

The present method can alternatively be carried out by the parenteraladministration, in doses of about 250 mg to about 2 g, of a synergisticantibacterial composition comprising 1 part by weight, as the diacid, ofthe 1-oxa compound I or a pharmaceutically acceptable salt thereof; andfrom about 0.02 to about 0.6 parts by weight of tobramycin, from about0.04 to about 2 parts by weight of amikacin, or from about 0.1 to about16 parts by weight of piperacillin. Such synergist antibacterialcompositions constitute additional embodiments of the present invention.The preparation of the compositions according to the present inventionmay be carried out by methods known in the pharmaceutical arts.

Dosages of the 1-oxa compound I in conjunction with tobramycin, amikacinor pipercillin, administered as synergistic antibacterial compositionsor as discrete quantities of the separate compounds can be repeated onceevery 4-12 hours until the infection is eliminated.

The biological activity (in vitro) of the antibiotic combinationsaccording to the present invention is demonstrated by the followingdescribed procedure.

EXPERIMENTAL METHOD FOR DETERMINING SYNERGY

Media used was Mueller-Hinton Broth (BBL) supplemented 20 mg Mg⁺⁺ /L and80 mg Ca⁺⁺ /L. Antibiotics used for this test were the 1-oxa compound I,tobramycin and pipercillin. A stock solution of pipercillin was preparedby dissolving a weighed amount of laboratory standard in pH 7 phosphatebuffer to give a concentration of 1000 ug/ml. Stock solution of compoundI was prepared by dissolving the contents of one vial of pre-weighed (10mg) laboratory standard in 10 ml of pH 7 phosphate buffer. Tobramycin issupplied as a solution containing 1 mg/ml. Working solution of theantibiotics was made by diluting the stock solutions with supplementedMueller-Hinton broth.

The checkerboard tube dilution technique was adapted to the microtitersystem using Autotiter plates containing 120 wells per plate. The platehas eight columns of 15 wells. The columns of the plates are designatedA through H and the rows of wells are numbered 1 through 15. Theaddition of the diluent (supplemented Mueller-Hinton broth containingantibiotic) and the two-fold serial dilution of antibiotics were doneusing an Autotiter V (Ames). With Autotiter system, it is possible todispense into the wells in each of the eight columns of the plate mediacontaining different concentrations of antibiotic (Antibiotic A). Eighttubes of media containing two-fold serial dilutions of antibiotic A wereprepared and placed in the dispensing jar of the multiple reagentdispenser. The antibiotic B to be tested in combination with theantibiotic A was placed in the antibiotic pickup tray in the Autotiter.All eight wells in the antibiotic pickup tray contain the sameconcentration of antibiotic B. Two-fold serial dilution of antibiotic Bwere made in the media containing antibiotic A. Two-fold dilutions ofantibiotic A were made in rows 1 through 13; row 14 contains mediawithout antibiotic and row 15 contains media plus antibiotic. Thediluted plate contains a two-fold serial dilution of antibiotic B inconstant concentration of antibiotic A (each column) in rows 1 through13 with the columns A through H containing a serial two-fold dilution ofantibiotic A in a decreasing concentration. The antibioticconcentrations made were chosen so that the range of concentration inthe diluted plate spanned the range of the minimum inhibitoryconcentrations (mic) of each individual antibiotic for each of theisolates tested. Fifty ul of diluent was dispensed into each well.

The inoculum was prepared from an overnight broth. The overnightcultures turbidity was adjusted to a MacFarland 0.5 turbidity standard(10⁸ cfu). The standard is prepared by adding 0.5 ml of 0.048M BaCl₂ to99.5 ml of 0.35N H₂ SO₄. Further dilution of the standardized overnightbroth culture is made to give a final concentration of 10⁵ colonyforming units. Fifty ul of this inoculum was added using 50 ul droppingpipettes to each of wells in columns A through H and rows 1 through 14.Row 14 contains no antibiotic and is used as a growth control; row 15 isnot inoculated and is used as a sterility control. The final volume ineach well in rows 1 through 14 is 100 ul. After inoculation, the platesare sealed with plastic tape and incubated at 35° C. for 18 to 24 hours.After incubation, the plates are read using an Autotiter viewer. Theturbid well containing only organism and media (row 14) is used forcomparison in determining growth end points.

The lowest concentration of antibiotic showing complete inhibition ofgrowth is the mic. The mic for the individual antibiotics for eachisolate being tested was confirmed at the time of testing forsynergistic combinations using a microdilution technique. The results ofthe synergy studies of combinations of the 1-oxa compound I andtobramycin or pipercillin against a number of strains ofmultiple-antibiotic-resistant Gram-negative organisms are summarized inTable II, while the results of synergy studies of 1-oxa compoundI-amikacin combinations are provided in Table III.

                                      TABLE II                                    __________________________________________________________________________    Results of Synergy Studies of Combinations                                    of the 1-Oxa Compound I and Tobramycin or Pipericillin                                                Combined                                                       I Alone                                                                            T* Alone                                                                           P* Alone                                                                           I   T   I   P                                         Organism (mcg/ml)                                                                           (mcg/ml)                                                                           (mcg/ml)                                                                           (mcg/ml)                                                                              (mcg/ml)                                      __________________________________________________________________________    Ps. aeruginosa                                                                1484     64   64    64  4   2   16  8                                         447      32   64   >128 <0.5                                                                              <0.5                                                                              4   0.5                                       424      8    64   >128 2   0.5 2   0.5                                       366      16   4    >128 0.5 2   8   16                                        84       16   8     64  2   1   4   4                                         Davis    >64  >64  >64  2   1   8   16                                        S. marcescens                                                                 100      0.25 16   >64  0.12                                                                              8   0.12                                                                              32                                        14142    1    >64  >64  0.25                                                                              4   0.12                                                                              8                                         302-2    1    16   >64  0.12                                                                              1   0.12                                                                              8                                         Townsend 4    32   >64  1   0.5 2   64                                        796      8    64   >64  2   16  2   4                                         E. cloacae                                                                    1253     4    32   >64  0.25                                                                              2   2   32                                        1415     0.12 32   > 64 0.12                                                                              <0.015                                                                            **                                            B293     0.5  >64  >64  0.12                                                                              8   0.25                                                                              16                                        1246     0.25 16   >64  <0.12                                                                             0.5 **                                            K. pneumoniae                                                                 214-1    2    32   >64  0.012                                                                             0.03                                                                              0.5 8                                         463      2    16   >64  0.012                                                                             <0.015                                                                            2   16                                        1204-2   2    >64  >64  0.012                                                                             0.03                                                                              0.5 2                                         512      0.5  >64  >64  0.012                                                                             0.06                                                                              0.5 32                                        933      0.25 32   >64  <0.012                                                                            0.5 **                                            130      0.5  16   >64  <0.012                                                                            0.5 **                                            P. rettgeri                                                                   65       0.25 64    64  0.12                                                                              1   **                                            P. mirabilis                                                                  Allen    0.06 32   >64  <0.12                                                                             1   **                                            P. Vulgaris                                                                   Shide    0.06 32   >64  <0.12                                                                             1   **                                            363      0.12 64   >64  <0.12                                                                             1   **                                            E. coli                                                                       Dill     0.25 >64   32  <0.12                                                                             4   **                                            1262     0.03 16   >64  <0.12                                                                             8   <0.12                                                                             64                                        136-3    0.06 64   >64  <0.12                                                                             4   **                                            Briley   0.12 4    >64  <0.12                                                                             8   <0.12                                                                             32                                        1415     **   **   **   <0.12                                                                             8   <0.12                                                                             2                                         46-2     0.03 >64  >64  <0.12                                                                             4   <0.12                                                                             2                                         1253     **   **   **   <0.12                                                                             4   0.5 4                                         1260     0.03 >64  >64  <0.12                                                                             0.5 **                                            77-3     0.03 64   >64  <0.12                                                                             0.25                                                                              **                                            Ps. maltophilia                                                               10       32   64   **   4   8                                                 505      8    32        0.6 0.5                                               11       4    4         0.5 2                                                 Acinetobacter sp.                                                             28-1     16   0.5  **   0.6 0.5                                               106      8    1         0.6 0.5                                               28-2     16   4         2   0.5                                               107      32   4         2   0.5                                               109      16   1         2   0.5                                               __________________________________________________________________________     *T = tobramycin                                                               P = pipercillin                                                               **Not tested                                                             

                  TABLE III                                                       ______________________________________                                        Results of Synergy Studies of Combinations                                    of the 1-Oxa Compound I and Amikacin                                                               Combined                                                 Organism   I* Alone  A* Alone  I      A                                       Isolate    (mcg/ml)  (mcg/ml)  (mcg/ml)                                       ______________________________________                                        P. rettgeri                                                                   768        0.5       16        <0.06  <0.5                                    53         0.25      8         <0.06  <0.5                                    997        0.4       8         <0.06  <0.5                                    1422       2         4         2      8                                       136-1      0.25      4         0.12   1                                       K. pneumoniae                                                                 938        0.25      4         <0.06  <0.5                                    130        0.5       4         0.5    <0.5                                    214-1      2         8         <0.06  <0.5                                    463        2         2         2      <0.5                                    1231       0.5       4         0.06   2                                       S. marcescens                                                                 ziegler    4         4         0.06   2                                       463-2      8         1         <0.06  <0.5                                    606        4         1         0.5    <0.5                                    B377       8         2         0.12   <0.5                                    1414       1         8         0.06   <0.5                                    E. cloacae                                                                    1253       4         1         <0.06  <0.5                                    1415       0.12      8         0.12   <0.5                                    228        0.12      2         <0.06  <0.5                                    1296       0.25      4         0.06   0.5                                     McQuinn    0.25      32        0.12   2                                       Ps. aeruginosa                                                                362        32        16        4      2                                       316-2      8         16        4      2                                       610        >64       1         0.6    1                                       363        32        32        1      2                                       Brownlee   32        16        1      4                                       Ps. maltophilia                                                               10         32        >64       8      <0.5                                    505        8         >64       2      1                                       Ps. pseudomallei                                                                         32        32        4      8                                       Acinetobacter sp.                                                             28-1       16        8         2      8                                       106        8         2         <0.06  <0.5                                    28-2       16        4         <0.06  <0.5                                    107        32        8         8      8                                       109        16        4         8      <0.5                                    ______________________________________                                         *A = amikacin                                                            

The results are expressed in terms of mic for each drug in thecombination. A four-fold decrease in concentration of each of the twocombined drugs from the concentration of the individual drugs requiredto obtain the specified effect is generally considered as acceptableevidence of synergism. However varying degrees of synergism can bedetected and expressed in terms of the Fractional InhibitoryConcentration (FIC) or the Fractional Bactericidal Concentration (FBC).The FIC, or the FBC, for each drug can be calculated by dividing theconcentration of each antibiotic present in the combination by theamount of each drug alone required for the same effect. Thus if A_(m)and B_(m) are the mics of drugs A and B alone against a given organism,and A_(c) and B_(c) are the mic's against that same organism of therespective drugs in the combination at the point of maximumeffectiveness, then ##EQU1## The degree of synergism exhibited isinversely proportional to the combination FIC (the sum of FIC_(a) andFIC_(b)). Where the FIC is less than 1 some synergy is indicated. Ofcourse, the smaller the combination FIC value the greater the degree ofsynergism indicated. As the FIC value approaches 1, a purely additiveeffect is indicated. An FIC of greater than 1 is indicative ofantagonism. A four-fold decrease in the mic of each drug in thecombination results in an FIC of 0.5.

The present invention is further illustrated by the following examples.

EXAMPLE 1

A patient suffering from a resistant Pseudomonas aeruginosa infection istreated by the sequential intravenous administration of 500 mg of the1-oxa compound I and 60 mg of tobramycin. Treatment is repeated every 8hours.

EXAMPLE 2

A patient suffering from an infection caused by Klebsiella pneumoniae istreated by administering intramuscularly 750 mg of the 1-oxa compoundand by intravenous infusion 1 g of piperacillin.

EXAMPLE 3

A patient suffering from a Pseudomonas aeruginosa infection is treatedby administering sequential intramuscular injections of sterilesolutions of 1 g of the 1-oxa compound and 1 g of piperacillin.Treatment is repeated about every 12 hours.

EXAMPLE 4

A patient suffering from an infection caused by Serratia marcescens istreated by the intravenous infusion of 250 mg of the 1-oxa compound I asa sterile solution of its disodium salt and concommitant intramuscularadministration of 90 mg of tobramycin sulfate.

EXAMPLE 5

100 Grams of the 1-oxa compound as its disodium salt and 200 grams ofpiperacillin are combined and blended. 1 Gram portions of the resultingcomposition are sealed in ampules.

EXAMPLE 6

A patient suffering from an infection caused by Pseudomonas aeruginosais treated by intramuscular administration of 1 g of the composition ofExample 5 in a suitable sterile diluent.

EXAMPLE 7

50 Grams of the 1-oxa compound as its disodium salt and 10 grams oftobramycin sulfate are combined and blended. 1 Gram portions of theresulting composition are sealed in ampules.

EXAMPLE 8

A patient suffering from an infection caused by Pseudomonas aeruginosais treated by intramuscular administration of 1 g of the composition ofExample 7 in a suitable sterile diluent.

EXAMPLE 9

A patient suffering from an infection caused by Serratia marcescens istreated by the sequential intramuscular administration of 250 mg. of the1-oxa compound I as its disodium salt in a sterile solution and 150 mg.of amikacin sulfate in a sterile solution. Treatment is repeated every 6hours.

EXAMPLE 10

A patient suffering from an infection caused by Klebsiella pneumoniae istreated by concommitant intravenous infusion of separate sterilesolutions of 350 mg. of the disodium salt of the 1-oxa compound I and100 mg. of amikacin sulfate. Treatment is repeated every 8 hours.

EXAMPLE 11

500 Grams of the 1-oxa compound I as its disodium salt and 100 grams ofamikacin sulfate are combined and blended. 1-Gram portions of theresulting composition are sealed in sterile ampules.

EXAMPLE 12

A patient suffering from an infection caused by Pseudomonas aeruginosais treated by intramuscular administration of 1 gram of the compositionof Example 11 in a suitable sterile diluent. Treatment is repeated every8 hours.

I claim:
 1. A method of treating infections caused bymultiple-antibiotic-resistant gram-negative organisms in a warm-bloodedanimal which comprises administering to said animal a 1-oxa antibioticcompound of the formula ##STR3## or a pharmaceutically acceptable saltthereof in an amount sufficient to produce blood levels of said compoundin said animal of at least about 0.12 to about 16 mcg/ml; andadministering in conjunction with said 1-oxa antibiotic amikacin in anamount sufficient to produce blood levels of amikacin in said animal ofbetween about 0.5 mcg/ml and about 8 mcg/ml.
 2. The method of claim 1wherein the multiple-antibiotic-resistant gram-negative organism isselected from the group consisting of Proteus rettgeri, Klebsiellapnemmoniae, Serratia marcescens, E. cloacae, Pseudomonas aeruginosa,Pseudomonas maltophilia, Pseudomonas pseudomallei and Acinetobacter. 3.A method according to claim 2 for the treatment of infections ofSerratia marcescens.
 4. The method of claim 1 wherein said 1-oxaantibiotic blood levels are produced by administering between about 250mg to about 2 g of the 1-oxa compound.
 5. The method of claim 4 whereinsaid amikacin blood levels are produced by administering between about 1mg/kg to about 6 mg/kg of amikacin.
 6. The method of claim 4 wherein thedosage of amikacin is about 1 mg/kg to about 3 mg/kg.
 7. A synergisticantibacterial composition comprising 1 part by weight, as the diacid, ofa 1-oxa compound of the formula ##STR4## or a pharmaceuticallyacceptable salt thereof and from about 0.04 to about 2 parts by weightof amikacin.
 8. A method of treating infections caused bymultiple-antibiotic-resistant gram-negative organisms in a warm-bloodedanimal which comprises administering to said animal an antibioticallyeffective amount of a composition of claim 7.